Shiro Amano

VEGF164-mediated Inflammation Is Required for Pathological, but Not Physiological, Ischemia-induced Retinal Neovascularization

Hypoxia-induced VEGF governs both physiological retinal vascular development and pathological retinal neovascularization. In the current paper, the mechanisms of physiological and pathological neovascularization are compared and contrasted. During pathological neovascularization, both the absolute and relative expression levels for VEGF164 increased to a greater degree than during physiological neovascularization. Furthermore, extensive leukocyte adhesion was observed at the leading edge of pathological, but not physiological, neovascularization. When a VEGF164-specific neutralizing aptamer was administered, it potently suppressed the leukocyte adhesion and pathological neovascularization, whereas it had little or no effect on physiological neovascularization. In parallel experiments, genetically altered VEGF164-deficient (VEGF120/188) mice exhibited no difference in physiological neovascularization when compared with wild-type (VEGF+/+) controls. In contrast, administration of a VEGFR-1/Fc fusion protein, which blocks all VEGF isoforms, led to significant suppression of both pathological and physiological neovascularization. In addition, the targeted inactivation of monocyte lineage cells with clodronate-liposomes led to the suppression of pathological neovascularization. Conversely, the blockade of T lymphocyte–mediated immune responses with an anti-CD2 antibody exacerbated pathological neovascularization. These data highlight important molecular and cellular differences between physiological and pathological retinal neovascularization. During pathological neovascularization, VEGF164 selectively induces inflammation and cellular immunity. These processes provide positive and negative angiogenic regulation, respectively. Together, new therapeutic approaches for selectively targeting pathological, but not physiological, retinal neovascularization are outlined.

Advanced glycation end products increase retinal vascular endothelial growth factor expression.

Advanced glycation end products (AGEs) are linked with the development of diabetic retinopathy; however, the pathogenic mechanisms are poorly defined. Vascular endothelial growth factor (VEGF) levels are increased in ischemic and nonischemic diabetic retina, and VEGF is required for the development of retinal and iris neovascularization. Moreover, VEGF alone can induce much of the concomitant pathology of diabetic retinopathy. In this study, we found that AGEs increased VEGF mRNA levels in the ganglion, inner nuclear, and retinal pigment epithelial (RPE) cell layers of the rat retina. In vitro, AGEs increased VEGF mRNA and secreted protein in human RPE and bovine vascular smooth muscle cells. The AGE-induced increases in VEGF expression were dose- and time-dependent, inhibited by antioxidants, and additive with hypoxia. Use of an anti-VEGF antibody blocked the capillary endothelial cell proliferation induced by the conditioned media of AGE-treated cells. AGEs may participate in the pathogenesis of diabetic retinopathy through their ability to increase retinal VEGF gene expression.

Alternatively spliced vascular endothelial growth factor receptor-2 is an essential endogenous inhibitor of lymphatic vessel growth

Disruption of the precise balance of positive and negative molecular regulators of blood and lymphatic vessel growth can lead to myriad diseases. Although dozens of natural inhibitors of hemangiogenesis have been identified, an endogenous selective inhibitor of lymphatic vessel growth has not to our knowledge been previously described. We report the existence of a splice variant of the gene encoding vascular endothelial growth factor receptor-2 (Vegfr-2) that encodes a secreted form of the protein, designated soluble Vegfr-2 (sVegfr-2), that inhibits developmental and reparative lymphangiogenesis by blocking Vegf-c function. Tissue-specific loss of sVegfr-2 in mice induced, at birth, spontaneous lymphatic invasion of the normally alymphatic cornea and hyperplasia of skin lymphatics without affecting blood vasculature. Administration of sVegfr-2 inhibited lymphangiogenesis but not hemangiogenesis induced by corneal suture injury or transplantation, enhanced corneal allograft survival and suppressed lymphangioma cellular proliferation. Naturally occurring sVegfr-2 thus acts as a molecular uncoupler of blood and lymphatic vessels; modulation of sVegfr-2 might have therapeutic effects in treating lymphatic vascular malformations, transplantation rejection and, potentially, tumor lymphangiogenesis and lymphedema (pages 993–994)

The International Workshop on Meibomian Gland Dysfunction: Report of the Diagnosis Subcommittee

Diagnostic tests of meibomian gland dysfunction (MGD) and of MGD-related disorders are based on the demonstration of abnormal anatomy and physiology of the glands and the detection of specific pathologic events. For this reason, this subcommittee report is divided into two sections. In part I, those aspects of meibomian anatomy and physiology that are relevant to currently available tests are described; a fuller account of the anatomy and physiology is provided in the report of the Anatomy Subcommittee of this workshop. In part II, each test and its performance is described in detail. In part III, the practical application of selected tests is summarized and recommendations for future approaches are made. Additional recommendations and a summary of pertinent literature and concepts are presented in Appendices 1 to 17.

Noncontact infrared meibography to document age-related changes of the meibomian glands in a normal population.

PURPOSE To examine the morphologic changes in meibomian glands associated with aging and gender using a novel meibography system and to assess their relation with slit-lamp findings regarding eyelid and tear film function in a normal population. DESIGN Cross-sectional observation case series. PARTICIPANTS Two hundred thirty-six healthy volunteers (114 men, 122 women; mean age+/-standard deviation, 41.2+/-23.1 years; range, 4-98 years). METHODS The upper and lower eyelids were turned over and the meibomian glands were observed using the noncontact meibography system, which consisted of a slit lamp equipped with an infrared charge-coupled device video camera and an infrared transmitting filter. A transilluminating light probe was not necessary. Partial or complete loss of the meibomian glands was scored for each eyelid from grade 0 (no loss of meibomian glands) through grade 3 (the lost area was more than two thirds of the total meibomian gland area). The tear film break-up time (BUT) was measured and tear film production was evaluated by Schirmer test. MAIN OUTCOME MEASURES Score of meibomian gland changes (meiboscore), tear film BUT, and Schirmer test value. RESULTS Using the meibography system, clear images of the meibomian glands were obtained in all subjects, including children. There were significant positive correlations between age and meiboscore in the entire subject population (R = 0.428; P<0.0001), as well as in males (R = 0.462; P<0.0001) and females (R = 0.418; P<0.0001). There were significant negative correlations between age and tear film BUT (R = -0.153; P = 0.019) and the Schirmer test value (R = -0.289; P<0.0001). The meiboscore was significantly positively correlated with the lid margin abnormality score (R = 0.359; P<0.0001). CONCLUSIONS The noncontact meibography system is a useful, quick, and patient-friendly method for obtaining information on the meibomian gland structure. Using this method, the authors found that changes in meibomian glands increase with age.

Changes in anterior and posterior corneal curvatures in keratoconus.

OBJECTIVE To quantitatively evaluate the changes in anterior and posterior corneal curvatures of eyes with keratoconus. DESIGN Case-control retrospective and observational study. PARTICIPANTS Thirty-one patients who were clinically diagnosed to have unilateral or bilateral keratoconus and 18 normal subjects. INTERVENTION The anterior and posterior topographies were obtained using scanning-slit videokeratography and assessed by Fourier series harmonic analysis. MAIN OUTCOME MEASURES Quantitative descriptors of the topography data, spherical power, regular astigmatism, and irregular astigmatism (asymmetry and higher order irregularity) components were compared between the anterior and posterior surfaces and among groups of clinically diagnosed keratoconus (33 eyes), keratoconus suspect (13 eyes), and normal subjects (36 eyes). RESULTS Spherical power (P = 0.0003, Mann-Whitney U test with Bonferronis correction of P values), regular astigmatism (P = 0.0166), and asymmetry (P = 0.0031) of the anterior surface were significantly greater in the keratoconus eyes than in the normal controls. For the posterior surface, spherical power (P<0.0001), regular astigmatism (P = 0.0143), asymmetry (P< 0.0001), and higher order irregularity (P = 0.0032) of the keratoconus group were significantly greater than those of the control group. The keratoconus suspect eyes, when compared with the normal controls, showed a significantly greater amount of spherical power (P = 0. 0166) and asymmetry (P<0.0001) in the anterior surface and spherical power (P <0.0001), regular astigmatism (P = 0.0244), asymmetry (P<0.0001), and higher order irregularity (P = 0.0276) in the posterior surface. All refractive components demonstrated statistically significant correlations between the anterior and posterior surfaces (P<0.0001, Spearmans rank correlation). CONCLUSIONS Not only the anterior but also the posterior corneal curvature is affected in keratoconus. These changes are observed from the early stage of this disorder.

Higher order wavefront aberrations of cornea and magnitude of refractive correction in laser in situ keratomileusis

OBJECTIVE To assess the relation between magnitude of refractive correction and changes in higher order wavefront aberrations of the cornea after laser in situ keratomileusis. DESIGN Prospective, consecutive, nonrandomized comparative trial (self-controlled). PARTICIPANTS One hundred eyes of 53 patients with myopia (-2.0 to -13.0 diopters) were included. INTERVENTION Laser in situ keratomileusis was performed. Videokeratography measurements were conducted before and 1 month after surgery. MAIN OUTCOME MEASURES The videokeratography data were used to calculate the higher order wavefront aberrations of the cornea for both small (3 mm) and large (6 mm) pupils. RESULTS For a 3-mm pupil, the surgery significantly increased coma-like (2.4 +/- 1.3-fold, P < 0.001, paired t test) and spherical-like (1.8 +/- 0.9-fold, P < 0.001) aberrations. For a 6-mm pupil, both coma-like (4.4 +/- 3.3-fold, P < 0.001) and spherical-like (9.4 +/- 5.2-fold, P < 0.001) aberrations were significantly increased by surgery. The amount of achieved correction showed significant correlations with the changes in coma-like (Pearson correlation coefficient r = 0.446, P < 0.001) and spherical-like (r = 0.348, P < 0.001) aberrations for a 3-mm pupil, and coma-like (r = 0.566, P < 0.001) and spherical-like (r = 0.693, P < 0.001) aberrations for a 6-mm pupil. The eyes that lost 2 or more lines of baseline spectacle-corrected visual acuity showed significantly larger induced increases in coma-like (P = 0.003, Mann-Whitney U test) and spherical-like (P = 0.009) aberrations for a 3-mm pupil than those that either improved or remained within 1 line of spectacle-corrected visual acuity CONCLUSIONS Laser in situ keratomileusis, performed using the current algorithms, increases higher order wavefront aberrations of the cornea, dependent on the amount of refractive correction.

Factors affecting the forward shift of posterior corneal surface after laser in situ keratomileusis

PURPOSE To evaluate the anteroposterior movement of the corneal back surface after laser in situ keratomileusis (LASIK). DESIGN Retrospective noncomparative case series. PARTICIPANTS One hundred ninety-six eyes of 120 subjects with myopic refractive errors of -2.0 to -12.5 diopters. INTERVENTION LASIK was performed. Corneal topography of the posterior corneal surface was obtained with the scanning slit topography system before and 1 month after surgery. MAIN OUTCOME MEASURES The amount of forward shift of the posterior corneal surface was determined. Multiple regression analysis was used to assess the factors that affect the forward shift of the posterior corneal surface. RESULTS After surgery, the posterior corneal surface displayed mean forward shift of 40.9 +/- 24.8 micrometer. Explanatory variables relevant to the forward shift of corneal posterior surface were, in the order of magnitude of influence, the amount of laser ablation (partial regression coefficient B = 0.561, P < 0.0001), preoperative corneal thickness (B = -0.176, P = 0.00096), and preoperative intraocular pressure (B = 1.676, P = 0.0053). Preoperative refraction and achieved myopic correction showed collinearity with the amount of laser ablation. CONCLUSIONS LASIK induces a forward shift of the cornea. Eyes with thinner corneas, higher intraocular pressure, and higher myopia requiring greater laser ablation are more predisposed to the anterior shift of the cornea.

Contact lens wear is associated with decrease of meibomian glands.

PURPOSE Approximately 30% to 50% of contact lens (CL) wearers report dry eye symptoms. Meibomian gland dysfunction has been recognized as a possible cause of CL-related dry eye. This study investigated the influence of CL wear on the meibomian glands using a newly developed meibographic technique. DESIGN Cross-sectional observational case series. PARTICIPANTS Contact lens wearers (n=121; 47 men, 74 women; mean age+/-standard deviation, 31.8+/-8.0 years) and healthy volunteers (n=137; 71 men, 66 women; mean age+/-standard deviation, 31.4+/-15.1 years). METHODS The following tests were performed: slit-lamp examinations of the eyelids, corneal and conjunctival staining using fluorescein, measurement of the tear film breakup time, evaluation of the meibomian glands using noncontact meibography, and measurement of tear production using the Schirmer I test. Partial or complete loss of the meibomian glands was scored for each eyelid using 4 grades (meiboscores): grade 0 (no loss of meibomian glands) through grade 3 (the area characterized by gland dropout was more than 66% of the total area containing the meibomian glands). The meiboscores for the upper and lower eyelids were summed for each subject. MAIN OUTCOME MEASURES Score of meibomian gland changes (meiboscore), tear film breakup time, and Schirmer test value. RESULTS The meiboscore was significantly higher (P<0.0001) in CL wearers (mean, 1.72; 95% confidence interval, 1.47-1.96) than in the control group (mean, 0.96; 95% confidence interval, 0.73-1.19). The average meiboscore of CL wearers was similar to that of a 60- to 69-year-old age group from the normal population. A significant positive correlation was observed between the duration of CL wear and the meiboscore. CONCLUSIONS Contact lens wear is associated with a decrease in the number of functional meibomian glands. This decrease is proportional to the duration of CL wear.

Corneal thickness measurements: scanning-slit corneal topography and noncontact specular microscopy versus ultrasonic pachymetry ☆

Purpose: To compare central corneal thickness measurements taken with 3 pachymetry systems: Orbscan scanning‐slit corneal topography/pachymetry, Topcon SP2000P noncontact specular microscopy, and Tomey ultrasonic pachymetry. Setting: Multicenter study, Tokyo, Japan. Methods: In 216 healthy eyes of 114 subjects, scanning‐slit topography, noncontact specular microscopy, and ultrasonic pachymetry were used in that sequence to record central corneal thickness. In another 20 healthy eyes of 13 subjects, 2 sets of measurements were repeated for each pachymetry to assess repeatability. Results: The mean central corneal thickness was compatible between scanning‐slit topography (546.9 &mgr;m ± 35.4 [SD]) and ultrasonic pachymetry (548.1 ± 33.0 &mgr;m); however, noncontact specular microscopy gave a significantly smaller mean (525.3 ± 31.4 &mgr;m) than the other 2 tests (P<.001, Tukey multiple comparison). There were significant linear correlations between scanning‐slit topography and noncontact specular microscopy (r = 0.846, P<.001), noncontact specular microscopy and ultrasonic pachymetry (r = 0.897, P<.001), and ultrasonic pachymetry and scanning‐slit topography (r = 0.852, P<.001). Noncontact specular microscopy tended to show the best repeatability; however, the difference was not statistically significant (P = .663, repeated‐measure analysis of variance). Conclusions: Corneal thickness readings were comparable between scanning‐slit topography and pachymetry; noncontact specular microscopy gave significantly smaller values. The measurements of the 3 methods showed significant linear correlations with one another. All methods provided acceptable repeatability of measurements.